The long term goal of our laboratory is to understand the mechanism coupling H+ translocation to catalytic activity in F1F0 ATP synthase. The enzyme serves the central metabolic role of energy production. A member of the F1F0 ATP synthase family is found in plants, animals and bacteria. The F1F0 ATP syntheses share a common molecular architecture and mechanism, so observations on one family member are generally applicable to all. In humans, defects in energy metabolism have been implicated in complex conditions including aging, Alzheimer's disease, cancer and diabetes. Distinct syndromes are attributed to specific inherited genetic defects in FIF0 ATP synthase. Therefore, a real need exists to understand the effects of mutations on the mechanism of the enzyme. The major goal of this proposal is to determine if the peripheral stalk of F1F0 ATP synthase plays a dynamic role in the mechanism of F1F0 ATP synthase. Specific Aim 1 studies the activity in purified F1F0 ATP synthases enzymes containing mutant b subunits in the peripheral stalk. These experiments will be supplemented with genetic studies to identify pairs of mutant uncF(b) genes capable of complementing each other. Specific Aim 2 looks at the positioning of the altered b subunits within the peripheral stalk. Specific Aim 3 determines if catalytic activity alters the orientation of the peripheral stalk. The experiments involve construction of F1F0 ATP synthase containing chimeric b subunits to allow determination of the relative positions of the two b subunits when the enzyme is at rest, inhibited or undergoing catalytic turnover. Specific Aim 4 considers the physical state of the stalk during catalysis. Probes of fluorescence environment will be positioned on one b subunit in the peripheral stalk and changes recorded while the enzyme is at rest or catalyzing ATP hydrolysis. A FRET strategy will be used to consider changes in the structure of FIFO complexes containing peripheral stalks of various lengths.